Molecular detection and identification of hemotropic Mycoplasma species in dogs and their ectoparasites in Iran

Hemotropic Mycoplasma species are vector-borne bacteria that attach and grow on the surface of erythrocytes in various mammals, yet reports of canine hemoplasmosis in Iran are scarce. The aim of this study was molecular detection and identification of hemoplasmas in the blood of dogs (n = 370) from five provinces of Iran and ectoparasites infesting them including Ctenocephalides canis and Pulex irritans fleas, Rhipicephalus sanguineus sensu lato ticks, Heterodoxus spiniger lice and Hippobosca longipennis keds. Hemotropic Mycoplasma spp. pathogens were detected using genus-specific conventional PCRs, and subsequently identified using species-specific PCRs for Mycoplasma haemocanis (Mhc), and Candidatus Mycoplasma haematoparvum (CMhp). Sanger sequencing was then performed to confirm the species. Correlation of infection and risk factors (geographical area, keeping condition, body condition, sex, age, ectoparasite infestation) were analyzed. In total, 210 dogs (56.7%) were tested PCR-positive for hemotropic Mycoplasma spp. Species-specific PCR and sequencing revealed infection with Mhc in 17.8%, with CMhp in 7.02% and co-infection in 31.9% of dogs. Flea infestation, poor body condition, and being older than 3-years-old correlated with hemoplasmosis. In ectoparasites, DNA of hemoplasmas were detected only in fleas i.e. Mhc in P. irritans, CMhp in P. irritans and C. canis, and co-infection in C. canis. To our knowledge, this is the first large-scale molecular epidemiology study of canine hemoplasmosis in Iran. Considering the high prevalence of canine hemoplasmosis all over the country including potentially zoonotic CMhp, effective ectoparasite control strategies, regular examination of dogs, successful chemoprophylaxis and public awareness strategies are advocated.

The prevalence of hemotropic Mycoplasma species in dogs has been reported to be between 1.2 23 and 54% 24 in different regions of the world.In particular, previous studies performed in the Middle Eastern countries e.g.Türkiye 22 , Qatar 25 , Saudi Arabia 26 and Egypt 27 reported prevalence of canine hemoplasmosis to be 38%, 7.8%, 5.7%, and 17% however, there is shortage of information about the occurrence and species composition of hemoplasmas in dogs of Iran since in the only two previous studies, limited number of animals from two cities (Shiraz and Esfahan) were examined 28,29 .Hence, this study aimed to assess the prevalence and molecular characterization of hemotropic Mycoplasma species in apparently healthy dogs and their ectoparasites from five Iranian provinces with different climates.

Hemoplasmas in ectoparasites
In total, 91 dogs (24.6%; 95% CI 19.5-29.2) were infested with ectoparasites which were identified as Ctenocephalides canis, Pulex irritans, Rhipicephalus sanguineus sensu lato, Heterodoxus spiniger.Out of 210 PCR-positive dogs, 52 (24.8%) were infested with ectoparasites at the time of sampling (Table 1).Fleas collected from four dogs in Hamedan scored PCR positive using universal primers.Further testing with species-specific PCRs showed DNA of Mhc in P. irritans, CMhp in P. irritans and C. canis, and DNA of pathogens in one C. canis specimen.Except for the dog from which the latter flea was collected, corresponding dogs were PCR-positive.None of the examined ticks were found infected.In addition to fleas, ticks and lice, 11 Hippobosca longipennis keds were collected from

Risk factors analysis for hemoplasma infection in dogs
Association between hemoplasma infection and potential risk factors are presented in Table 1.The presence of hemoplasma DNA was significantly higher in dogs older than 3-years-old.Furthermore, infection rate was higher in dogs with flea infestation and with poor body condition.There was no statistically significant association between positivity and gender or breed (Table 1).

Sequence and phylogenetic analyses
BLAST analysis of consensus sequences for detected pathogens displayed 99.8-100% nucleotide identity with Mhc and CMhp isolates available in GenBank® database.Representative sequences of pathogens detected in this study were deposited in the GenBank® database under the accession numbers OQ474934, OQ474935, OQ474936 for Mhc, OQ474937 for CMhp in dogs, OQ572680 for CMhp in C. canis and P. irritans, OQ572681 for Mhc in P. irritans.Molecular identification of nucleotide sequences for both organisms was supported by the distinct separation of species-specific clades inferred from the phylogenetic analyses (Fig. 1).

Discussion
The high frequency of canine hemoplasmosis (56.8%) reported herein, indicates that apparently-healthy dogs from different regions in Iran are exposed to Mhc and CMhp, which poses health risks not only to canine but also people in contact with them.To our knowledge, the molecular prevalence found in this study is the highest infection rate reported in dogs to date although considerably high prevalences were previously reported in Australia (54% 24 ), Brazil (44.7% 30 ), Sudan (42.3% 31 ) and, Portugal (40% 32 ).Although both hemoplasma species were recorded in the country, previous studies reported much lower prevalence i.e. 10% in Shiraz 33 , and 26% in Esfahan 28 which could potentially be due to environmental and climatic factors, veterinary care, and differences in the population structure (e.g.age) of the examined dogs.In particular, the infection was highest in dogs in Khuzestan (69.6%) and Mazandaran (66.7%) with hot-humid and Mediterranean climates, respectively, which favor the propagation of arthropod vectors.In neighboring Türkiye, 38% of sheltered dog 22 and, in another study 15.3% of a mixed population stray, sheltered and domestic dogs 34 were positive by PCR.In other studies in the region, 5.7% of stray dogs in Saudi Arabia were positive for Mhc 26 , 7.8% of client-owned dogs in Qatar 25 , and 17% of client-owned pet dogs in Egypt were positive for Mycoplasma spp. 27.Present and previous reports suggest that apparently-healthy dog populations in West Asia and North Africa are chronically infected with hemoplasmas suggesting some level of enzootic stability against these pathogens.Since hemotropic Mycoplasma spp.including CMhp herein detected in 38.9% of dogs can infect humans 2 , raising the awareness of the public, persons in close relation with dogs (e.g.owners and veterinary personnel) and physicians about the risk of acquiring the infection from dogs is necessary.Moreover, since 30,35 arthropods 36 are still considered major players in the epidemiology of hemoplasmas, routine control of fleas and ticks is advocated.Finally, test-and-treatment of dogs even in the absence of relevant symptoms are strongly recommended.
In western Asia and north Africa, little information is available about the composition of hemotropic Mycoplasma species and genogroups infecting dogs.We found a higher prevalence of Mhc (17.83%) in comparison with CMhp (7.02%).Since the first record of CMhp in a dog in southern city of Shiraz 33 few molecular-based studies have been performed.In Shiraz, 6 (6.0%) and 4 (4.0%) of 100 examined dogs were positive for Mhc and CMhp with no case of concurrent infection 29 .In another study in the same city, Mhc and CMhp were detected in 4 (7.5%) and 3 (5.7%)out of 53 dogs 37 .Similarly, examination of 100 dogs in central city of Esfahan showed higher prevalence of Mhc (13%) compared to CMhp (10%) 28 .In Türkiye also, infection of dogs with Mhc was higher than CMhp in two studies 22,34 .In contrast, CMhp was the dominant species infecting dogs in Egypt (15% vs 2% Mhc) 27 .However, it seems that co-infection with both species is common in dogs of the region i.e. 31.89% in this study, 3% in Esfahan Iran 28 , 5.3 and 6.4% in Türkiye 22,34 , and 2% in Egypt 27 suggesting co-transmission of the pathogens.
Detection of Mhc in P. irritans, CMhp in P. irritans and C. canis, and simultaneous presence of pathogens in one C. canis indicates potential role of these fleas in the transmission of both pathogens in Iran although detection of pathogens in the body of fleas could be due to bloodmeal intake.However, statistically significant association between the presence of hemoplasma DNA in dogs' blood and infestation with fleas, and simultaneous detection of hemoplasmas in three dogs and three fleas gives weight to the role of fleas in the transmission of hemoplasmas among dogs.The role of arthropod vectors in the epidemiology of hemoplasmosis is supported by the detection of canine and feline hemoplasma DNA in fleas collected from animals and/or the environment 1 .In particular, the cat flea, C. felis, has been implicated in feline hemoplasma transmission, but this has not been definitively proven in experimental studies 38 .Experimental studies on common flea species e.g. C. canis, C. felis, C. orientis and P. irritans could shed light on the competence of these fleas.
There are controversies regarding the role of ticks in the epidemiology of canine hemoplasmas.Only a single study has experimentally demonstrated transmission of Mhc by R. sanguineus s.l. 18, and the role of this tick in the natural transmission of Mhc and other hemoplasmas is unknown.In a study in Switzerland, bacterial DNA was detected in Ixodes spp.and Rhipicephalus spp.ticks collected from animals, but not in unfed questing Ixodes spp.collected from vegetation 20,39 .Furthermore, higher prevalences of canine hemoplasmosis have been reported in countries and regions where R. sanguineus s.l.ticks are found more commonly 40 hypothesizing that this tick may be a possible vector for the transmission of canine hemoplasma species.In contrast, none of 350 individual R. sanguineus ticks collected from dogs in a study in Türkiye where brown dog tick is prevalent 41 ; and none of the tick specimens including 204 fully engorged nymphs removed from dogs, and 2100 nymphs and 85 adults collected from the grounds of the same shelter where both pathogens were present in examined dogs suggest that R. sanguineus s.l. may not be a suitable vector for canine hemoplasma species in the field conditions 22 .In the latter study, hemoplasma DNA was not detected in unfed adult ticks molted from engorged nymphs that had been collected from hemoplasma-infected dogs indicating that transstadial transmission did not occur in this tick species 22 .Thus, the importance of ixodid ticks including R. sanguineus s.l. in the transmission of canine hemotropic mycoplasmas remains unclear.
Relatively high prevalence of hemoplasmosis in dogs of this survey compared to previous studies might be associated with the examined population i.e. 98.1% were sheltered animals.In Iran, the animal shelters are mainly built by charity-based non-governmental organizations (NGOs) to feed stray dogs which are rescued from all over the province and protect them from culling program.Unfortunately, other than below-standards sanitation of these shelters, density of dogs is very high and thus posing dogs to stress and tension (AS personal observation) which could potentially lead to behavioral changes and aggressive interactions.Studies have successfully transmitted feline hemoplasma infection via subcutaneous inoculation of blood containing low numbers of organisms, suggesting possibility of pathogens transmission by fighting and biting in the field 42 .Interestingly, in a study in Cambodia transmission of haemotropic Mycoplasma was shown in the total absence of arthropod vectors in a closed population of dogs on ectoparasiticides 43 .In that study forty dogs were treated with two ectoparasiticide products and monitored for eight months.No new infections caused by Babesia vogeli, Ehrlichia canis, Anaplasma platys, and Hepatozoon canis which are proven to be vectorially-transmitted were detected; conversely, the number of haemoplasma infections in dogs rose significantly, providing strong evidence of non-vectorial transmission.Authors reported frequent dog aggression and fighting 43 similar to the situation in Iran shelters.Other than possible transmission of hemoplasmas in fighting-when as little as 10 μl of cat blood infected with Candidatus Mycoplasma turicensis can produce an infection in a naïve cat 44 -DNA of C. M. turicensis was detected in the saliva and feces 20 , and DNA of C. M. haemominutum was detected in the salivary glands of infected cats 45 .Specific research on the transmission of canine haemoplasmas is crucial for establishment and implementation of methods to prevent new patients.
Association of infection rate and higher age of dogs is in agreement with the recent studies, which reported hemoplasmas to be more prevalent in older dogs 26,40,46 and could be due to longer exposure time to pathogen.However, we found no significant association between hemoplasma infection rate and gender or breed, which is in accordance with previous reports in Cuba 47 , Nigeria 48 , Türkiye 34 and Mediterranean countries 32 .Moreover, we found a significant association between hemoplasma positivity and body condition score of dogs i.e. 69.6% of dogs with poor body condition were PCR positive.This finding is somehow expectable since general health status of dogs is known to be linked with hemoplasmosis 34,49,50 .
There were some limitations in the present study such as limited number of pet dogs examined and number of Sanger sequenced samples because of resources.Moreover, hematological analyses could provide insightful information about the pathogenicity of strains since dogs in this study were asymptomatic.Additionally, future studies would benefit from typing the isolates using a panel of gene loci to shed light on possible differences in virulence and geographical distribution of strains.Finally, application of more sensitive and quantitative www.nature.com/scientificreports/diagnostic methods e.g.real-time PCR will be beneficial not only in detecting more positive dogs but also to discover possible relationship between bacterial load and clinical manifestation.

Conclusion
To our knowledge, this is the first large-scale molecular epidemiology study of canine hemoplasmosis in Iran.
Considering the high prevalence of hemoplasmas in dogs all over the country including potentially zoonotic CMhp, routine ectoparasites' control and test-and-treatment strategy especially prior to adopting sheltered dogs are recommended.Raising the awareness of the public, persons in close relation with dogs such as owners, shelters' personnel and veterinary professionals, as well as physicians about the risk of acquiring the infection from dogs is also necessary.Last but not least, since recent findings suggest the transmission of canine haemoplasmas without involvement of arthropod vectors, establishment and implementation of novel methods to prevent their transmission is very much needed.

Ethical aspects
All applicable international, national, and institutional guidelines for the care and use of animals were followed.

DNA extraction
Genomic DNA was extracted from 200 µl of the blood samples using FavorPrep™ Genomic DNA Extraction Mini Kit (Favorgen, Pingtung, Taiwan) and from ectoparasites (ticks, fleas, keds) using Cell/Tissue WizPrep™ gDNA

Molecular detections of the hemoplasma species
DNAs were initially screened by universal Mycoplasma spp.primers that amplifies a fragment of 16S rRNA gene 53 .Positive samples were further subjected to two species-specific PCR assays to determine the presence of CMhp and Mhc DNA using species-specific primers 54 .Primers sequences, expected amplicon sizes, and thermal cycling conditions are given in PCR amplified products were analyzed in 1% (w/v) agarose gel (SinaClon, Iran) stained with 0.5 µg/ml ethidium bromide (SinaClon, Iran) and electrophoresed at 110 V for 55 min.The gels were visualized and photographed using UV Camera and transilluminator (Vilber Lourmat, Collégien, France).

Sequencing and phylogenetic analysis
Positive amplicons of Mycoplasma spp.from dog blood (n = 4) and fleas (n = 2) were Sanger sequenced in an Applied Biosystems 3500 Genetic Analyzer (Thermo Fisher Scientific, MA, USA) by Pishgam Biotech Company (Tehran, Iran).The sequencing results were first analyzed using Local Basic Alignment Tool (BLAST), edited by SnapGene ® software (GSL Biotech LLC, Chicago, USA), and then submitted to GenBank ® (NCBI) (www.ncbi.nlm.nih.gov).Alignments of obtained 16S rRNA sequence compared to the closely related sequences found in the GenBank ® database and phylogenetic tree construction were performed using Mega X (Molecular Evolutionary Genetics Analysis version 10) 55 , based on maximum likelihood method 56 .Evolutionary analyses were conducted on 1000 bootstrap replications.

Statistical analyses
Association between the prevalence of canine hemoplasmas and potential risk factors i.e. age, breed, gender, sampling location, living condition, co-infections, and BCS of dogs were analyzed using a chi-square and Fisher's exact test by SPSS version 26.0.(IBM Corp, Armonk, NY).P-values < 0.05 were considered as statistically significant differences.

Figure 1 .
Figure 1.Phylogenetic relationship of Mycoplasma haemocanis (marked with circle) and Candidatus Mycoplasma haematoparvum (marked with triangle) sequences isolated in this study to other Mycoplasma spp.based on a partial sequence of the 16S rRNA gene.The analyses were performed using maximum likelihood method.Homologous sequence from Mycoplasma pneumonia ATCC29342 (accession number: NR077056) was used as the outgroup.Mhc indicates the Mycoplasma haemocanis species and CMhp indicates Candidatus Mycoplasma haematoparvum species.

From
December 2018 to February 2021, cephalic or saphenous vein blood specimens were collected from 370 dogs in five provinces of Iran with different climates i.e.Hamedan (n = 135; cold semi-arid) and Kermanshah (n = 50; warm and temperate) in the west, Yazd (n = 74; hot and arid) in the center, Khuzestan (n = 69; hot and humid) in the south-west and Mazandaran (n = 42; mild and humid) in the north (Fig.2).Animal data i.e., gender, breed, age, keeping condition, body condition score and ectoparasites infestation were recorded.Blood and ectoparasite specimens were transferred to the Microbiology Laboratory of Faculty of Veterinary Medicine, Bu-Ali Sina University.Blood samples were kept in − 70 °C freezer until DNA extraction.Collected ectoparasites were counted and identified to species level using taxonomic keys51,52 at the earliest convenience then stored in separate microtubes in − 70 °C freezer until further molecular examination.

Figure 2 .
Figure 2. Samples were collected from five provinces in Iran.The map was drawn by using ArcGIS software version 10.3 (https:// enter prise.arcgis.com/ en/ portal/).

Table 1 .
Risk factors associated with prevalence of Mycoplasma haemocanis (Mhc) and Candidatus M.
The blood and ectoparasites of dogs were collected with permission of the Ethical Committee of Hamadan University of Medical Sciences, Iran (code: IR.UMSHA.REC.1398.124)and the Ethical Committee of Bu-Ali Sina University, Iran (codes: IR.BASU.REC.1399.0014and IR.BASU.REC.1399.0021).All methods reported in the present study are in accordance with ARRIVE guidelines (https:// arriv eguid elines.org).
Mini Kit (Wizbiosolutions, Seongnam, South Korea).If more than one specimen belonging to one ectoparasite species was collected from a dog, specimens were pooled prior to DNA extraction.Quantity (260 nm absorbance) and quality (260/280 nm absorbance ratio) of extracted DNAs were measured by NanoDrop spectrophotometer (Thermo Scientific™ NanoDrop 2000, Waltham, MA, USA).DNAs were stored at − 20 °C until PCR screening.

Table 2 .
PCRs were performed in a 25 µl volume reaction mixture consisting of 8 µl of distilled deionized water, 12.50 µl of Taq DNA Polymerase 2× Mastermix, 2.50 µl of the template DNA, and 1 µl of each forward and reverse primer.The concentration of primer was 10 pmol, except for CMhp-F and CMhp-R (30 pmol).PCR amplifications were performed in a SimpliAmp TM thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA).Positive DNA controls (CMhp & Mhc) that were kindly provided by Professor Dr. Roberta Iatta (University of Bari Aldo Moro, Italy), and distilled deionized water were used as positive and negative controls in each run.

Table 2 .
The oligonucleotide primers and PCR conditions used for molecular detection of the hemoplasma species.